There is a requirement to isolate DNA rapidly and conveniently from a variety of cellular sources, including blood. The availability of DNA has greatly facilitated the analysis and characterisation of the genome in many organisms through the application of sequencing and hybridisation techniques. Conventional approaches to DNA isolation and purification are based on multi-step procedures involving phenol/chloroform (see for example Sambrook, J. et al, Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring harbor Laboratory Press, 1989). These processes are inherently laborious, may result in damaged DNA samples and are generally not amenable to automation. A number of non-toxic extraction procedures have been reported (Nucleic Acids Research, 15, 859, 1987; Analytical Biochemistry, 120, 282-288 1982), but these require either extensive dialysis or use of filters. Improved extraction methods include the use of chaotropic agents (BioTechniques, 22, 550-553, 1997). Others may be applicable to specific cell types and involve only lysis, dilution and addition to a PCR tube (BioTechniques, 11, 30-31, 1991).
U.S. Pat. No. 5,650,506 (Becton Dickinson) relates to modified glass fiber membranes which exhibit sufficient hydrophilicity and electropositivity to bind DNA from a suspension containing DNA and permit elution of the DNA from the membrane. The modified glass fiber membranes are useful for purification of DNA from other cellular components. A product is also available based on isolation of DNA from blood on glass filters (GFX™ Genomic Blood DNA Purification Kit, Amersham Pharmacia Biotech).
U.S. Pat. Nos. 5,705,628 and 5,898,071 disclose a method for separating polynucleotides, such as DNA, RNA and PNA, from a solution containing polynucleotides by reversibly and non-specifically binding the polynucleotides to a solid surface, such as a magnetic microparticle. A similar approach has been used in a product, “Dynabeads DNA Direct” marketed by Dynal A/S, Norway.
U.S. Pat. No. 5,447,864 discloses a method of separating cell nuclei from cells by means of a pipette tip device, open at one end and having a membrane extending across its forward end. The method comprises treating a fluid containing whole cells so as to selectively lyse the cytoplasmic membrane, together with a small proportion of the nuclear membranes, but leaving a large proportion of the cell nuclei intact. The treated fluid is applied to the membrane whereby a mesh of DNA from the lysed nuclei is formed on the surface and captures intact cell nuclei. The mesh comprising DNA on the surface is then washed to separate the captured cell nuclei from other components of the cells. A device for use in the method is also described, the device comprising a pipette tip having a membrane that extends across its forward end.
Methods for the isolation of DNA in microstructured devices have demanded substantial simplification of conventional techniques that are time-consuming and frequently require centrifugation, pipetting, vortexing or thermal incubation steps. One approach to the purification of DNA from whole blood is to isolate the white blood cells prior to direct PCR (Nucleic Acids Research, 24, 380-385, 1996), thus removing a primary inhibitor of PCR, namely haemoglobin. Another approach (Science, 282, 399-401, 1998) involves mixing blood with a salt solution that lyses the cells. The lysate is then introduced into a chamber containing a glass wall on which DNA binds by charge interaction, while the rest of the sample is ejected. The DNA is washed with ethanol/water mixes and then eluted to a neighbouring chamber.
WO 97/21090 relates to methods and apparatus for performing micro-analytic and micro-synthetic procedures. The invention provides an apparatus comprising a rotatable disc which includes sample inlet port, fluid micro-channels, reaction chambers and outlet ports. Movement of fluids within the device, for example reagents, samples and other liquid components, is facilitated by rotation of the disc causing centripetal acceleration of the fluids through the micro-channels embedded in the disc. Methods specific for the apparatus are provided for performing a variety of procedures, including DNA synthesis, micro-extraction and cell counting.
A method for the extraction and concentration of short (500 bp) and medium size (48000 bp) DNA from test samples of bacteriophage lambda DNA utilising silicon fluidic microchips is disclosed in J.Biomechanical Engineering, 121, 23-27 (1999). PCR and gel electrophoresis were used to analyse the nucleic acid obtained by this process.